Transgenic plants expressing cobalamin binding proteins

ABSTRACT

The present invention relates to the use of transgenic plants for the expression of vitamin B12 (cobalamin) binding proteins. Plant cells are transformed with nucleotide sequences adapted for expression and secretion of vitamin B12 binding proteins. The present invention also relates to the use of recombinant vitamin B12 binding proteins from plants in analytical tests and treatment of vitamin B12 deficiency. Also disclosed is a method for purification of recombinant vitamin B12 binding proteins.

[0001] The present invention generally relates to transgenic plantproduction of recombinant proteins which are able to bind cobalamin. Therecombinant proteins may be used 1) prophylactically and/ortherapeutically to treat cobalamin deficiency and 2) analytically.

[0002] Two groups of proteins are characterized by specific binding tovitamin B12, (cobalamin), hereafter called cobalamin binding proteins(CBP). One group consists of proteins involved in the assimilation ofcobalamin and the other group is involved in the metabolism andutilization of cobalamin and includes the enzymes using cobalamin as acofactor. Human assimilation of dietary cobalamin (Cbl) is a complexprocess with three successive Cbl-transporters involved: haptocorrin(HC), intrinsic factor (IF) and transcobalamin (TC) (1-4). Theseproteins strongly bind to Cbl which is synthesized by bacteria. Thethree CBPs provide uptake of the Cbl from the food to the body. IF isthe main binder of Cbl in the intestinal tract and the IF-Cbl complexbinds to an intestinal receptor resulting in the internalization of theCbl to the blood (3, 4). In the blood the TC-Cbl complex distributesassimilated Cbl among the tissues where it binds to one or more specificreceptors present in the cell membrane (2, 4). A significant amount ofCbl circulates in blood bound to HC (1, 4). The exact function of HC isnot identified. Inherited HC deficiency does not provoke any visiblepathological effect (5) in contrast to the cases of IF- andTC-deficiencies (6, 7).

[0003] Two Cbl dependent enzymatic reactions have been identified inmammals. The conversion of methylmalonyl-CoA to succinyl-CoA involvesadenosyl-Cbl (Ado-Cbl) as a cofactor. Methyl-Cbl is a cofactor in thesynthesis of methionine from homocysteine. Prior to the presentinvention, only small amounts of CBP have been isolated. Purification ofCbl binders is complicated by low concentration of these proteins innatural sources (1-4). Isolation of 1 mg of TC requires, for instance,150-300 liters of human plasma (8, 9). Human IF can be isolated fromstomach juice which contain about 1 mg IF per liter. Isolation of pureTC and IF from natural sources is also complicated by the presence ofrelatively large amounts of HC in these sources. An expression systemhas been established for IF and TC in insect cells (10, 11) andrecombinant proteins were obtained at the level of 10-100 μg. We haveexpressed human and bovine TC in yeast and obtained 5-7 mg recombinantprotein from a total of 1 liter of fermentation media (12). Human IF hasbeen expressed in yeast and the yield was in the range of 1-4 mg perliter of total fermentation media (13). These expression systems areexpensive to use and they deliver a mixture of holo- and apo-forms ofthe CBP. This is also the case for natural sources. The holo-form is theCBP in complex with Cbl, whereas the apo form is a CBP not complexed toCbl. For use in diagnostic kits and other analytical purposes it isimportant to achieve purely either the apo- or holo-form of CBP.

[0004] IF isolated from human gastric juice is used for diagnosticpurposes in the Schilling test where the patient ingests the isolatedIF. Therefore, there is a risk for transmission of human diseases fromthe donor to the patient. Similarly, the use of IF isolated from pig orrecombinant human IF isolated from the yeast expression media whichcontains an enzymatic hydrolysate of meat, may cause a risk fortransmission of animal diseases to the patient. The use of recombinanthuman IF from a plant expression system will eliminate these problems.

[0005] Plants do not contain Cbl or CBPs. Therefore, recombinant plantswith an inserted gene for i.e. human IF will express only the apo-formof IF. This makes plants very suitable as a “factory” for production ofthe apo-form of CBPs with no contamination of the holo-form. In othereukaryotic expression systems the recombinant CBP will be more or lesssaturated with Cbl resulting in a mixture of holo- and apo-form CBP. Allother eukaryotic organisms except plants use Cbl in enzymatic reactionsand therefore contain Cbl. Similarly, the use of transgenic plantsresults in recombinant CBP free of other CBPs since plants do notnaturally express CBP. Isolation of IF from other sources, i.e. gastricjuice results in a preparation contaminated with some HC since both CBPsare present in gastric juice.

[0006] CBPs expressed in plants but not other organisms are only in theapo-form and therefore the plant expression system gives the opportunityfor purification of the CBP by affinity column chromatography with acobalamin column. The holo-form of CBP does not bind to the cobalamincolumn. Therefore, CBPs expressed in plants are easier to purify thanCBPs expressed in other organisms.

[0007] CBPs expressed in plants can be designed to have or alternativelynot to have identical amino acid sequences as their corresponding nativeproteins. Posttranslational modifications such as disulphide bondformation between cystein residues will be present in the recombinantproteins. For CBPs with amino acid sequences identical with native CBPsthe localization of disulphide bonds are expected to be identical, sinceCBPs from plants binds Cbl. Therefore the tertiary structure ofrecombinant CBPs are expected to be identical to native CBPs. Thespecific binding of recombinant human IF (rhIF) from plants in complexwith Cbl to the intestinal receptor cubilin support an identicaltertiary structure between native hIF and rhIF from plant. Theglycosylation of recombinant CBPs from plants will not be identical tothe glycosylation of the corresponding native CPBs, since glycosylationis cell type specific. Therefore, rhIF from plants differ from native IFby the composition of the sugars in the carbohydrate chains.

[0008] plant” is a plant where some or all of its cells contain anexpression vector or a fragment of an expression vector.

[0009] In a first preferred embodiment the protein capable of bindingcobalamin or analogs thereof would be any one of transcobalamin,intrinsic factor, haptocorrin, methylmalonyl-CoA mutase, methioninesynthase or a protein with at least 60% identity to any one of theseproteins or a fragment thereof. The percent identity of two amino acidsequences or of two nucleic acid sequences is determined by aligning thesequences for optimal comparison purposes (e.g., gaps can be introducedin both sequences for best alignment) and comparing the amino acidresidues or nucleotides at corresponding positions. The “best alignment”is an alignment of two sequences which results in the highest percentidentity. The percent identity is determined by the number of identicalamino acid residues or nucleotides in the sequences being compared(i.e., % identity=# of identical positions/total # of positions×100).

[0010] The determination of percent identity between two sequences canbe accomplished using a mathematical algorithm known to those of skillin the art. An example of a mathematical algorithm for comparing twosequences is the algorithm of Karlin and Altschul (1990) Proc. Natl.Acad. Sci. USA 87:2264-2268, modified as in Karlin and Altschul (1993)Proc. Natl. Acad. Sci. USA 90:5873-5877. The NBLAST and XBLAST programsof Altschul, et al. (1990) J. Mol. Biol. 215:403-410 have incorporatedsuch an algorithm. BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilised as described in Altschul et al.(1997) Nucleic Acids Res. 25:3389-3402. Alternatively, PSI-Blast can beused to perform an iterated search which detects distant relationshipsbetween molecules (Id.). When utilising BLAST, Gapped BLAST, andPSI-Blast programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0011] Another example of a mathematical algorithm utilised for thecomparison of sequences is the algorithm of Myers and Miller, CABIOS(1989). The ALIGN program (version 2.0) which is part of the CGCsequence alignment software package has incorporated such an algorithm.Other algorithms for sequence analysis known in the art include ADVANCEand ADAM as described in Torellis and Robotti (1994) Conput. Appl.Biosci., 10:3-5; and FASTA described in Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-8. Within FASTA, ktup is a control option thatsets the sensitivity and speed of the search.

[0012] However, the important factor is that a protein with a similarsequence identity to natural CBP is also functionally the same as theCBP binding proteins. In other words, the proteins produced according tothe present invention are able to make a complex with Cbl and bind tothe corresponding CBP receptor.

[0013] The plant is preferably a “Generally Recognised As Safe” (GRAS)plant. Plants that only produce pollen in the second year of the lifecycle such as carrots can be used to prevent cross contamination ofother plants in the environment with genetically modified material.

[0014] A whole plant can be regenerated from the single transformedplant cell by procedures well known in the art. The invention alsoprovides for propagating material or a seed comprising a cell. Theinvention also relates to any plant or part thereof includingpropagating material or a seed derived from any aspect of the invention.

[0015] In a second aspect the present invention provides a cobalaminbinding protein that is expressed in a transgenic plant, or bytransgenic plant cell culture. This protein is preferably isolated orpurified from the plant material. This protein can be either in the apo-(i.e. not bound to cobalamin) or holo-form (i.e. bound to cobalamin.)The protein that is produced by the transgenic plant has differentcarbohydrate moieties attached to the molecule compared to a similarprotein produced by humans or micro-organisms such as yeast. Thevariation in the carbohydrate content of the proteins can be seen by thedifferent apparent molecular weights of the protein produced by thetransgenic plant as compared to the protein produced by humans or yeast.Once the proteins are stripped of the carbohydrate groups, all theproteins have the same molecular weight as seen by SDS-Polyacrylamidegel electrophoresis.

[0016] In a third aspect the present invention provides a method ofisolating and/or purifying the cobalamin binding protein or a functionalfragment thereof as defined above comprising the following steps:

[0017] (a) Homogenisation of the transgenic plant material;

[0018] (b) Filtration of the supernatant formed by centrifugation of thehomogenate;

[0019] (c) Affinity column chromatography using cobalamin

[0020] (d) Elution of the cobalamin binding protein attached tocobalamin

[0021] (e) Gel filtration;

[0022] And optionally further comprising the following steps to producethe apo-form of the cobalamin binding protein:

[0023] (f) Dialysis against 5M guanidine HCL;

[0024] (g) Dialysis against 0.2M sodium phosphate.

[0025] In a fourth aspect the present invention provides a compositioncomprising a cobalamin binding protein of the present invention. Thecobalamin binding protein can be provided in a substantially pure form.Alternatively, it can be provided as transgenic plant material which israw, unprocessed transgenic plant material or more or less processedtransgenic plant material that is ingested e.g. in the form of chopped,ground, homogenized transgenic plant material, liquid transgenic plantextract as well as partially or completely purified recombinant CBP.

[0026] The composition is preferably a pharmaceutical composition. Thecompositions of the invention may be adapted for administration by anyappropriate route, for example by the oral (including buccal orsublingual) route. Such formulations may be prepared by any method knownin the art of pharmacy, for example by bringing into association theactive ingredient with the carrier(s) or excipient(s).

[0027] Pharmaceutical formulations adapted for oral administration maybe presented as discrete units such as capsules or tablets; powders orgranules; solutions or suspensions in aqueous or non-aqueous liquids;edible foams or whips; or oil-in-water liquid emulsions or water-in-oilliquid emulsions.

[0028] Preferred unit dosage formulations are those containing a dailydose or sub-dose, as herein above recited, or an appropriate fractionthereof, of an active ingredient.

[0029] It should be understood that in addition to the ingredientsparticularly mentioned above, the formulations may also include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavouring agents. The recombinant plant CBP may also bedelivered in combination with a second component like cobalamin orcobalamin analogs or cobalamin containing a tracer atom (or molecule).Tablets with recombinant plant CBP with or without a second componentmay also be designed for the release of CBP in the intestine to avoidthe low gastric pH and the gastric proteases.

[0030] Preferably, the compositions of the invention are presented fororal use. The recommended daily dose for vitamin B12 is 2.5 microgramswhich corresponds to 125 micrograms of IF-Cbl or 122.5 micrograms of IF.

[0031] In a fifth aspect the present invention provides a foodstuffcomprising transgenic plants of the invention or plant material derivedfrom such plants.

[0032] In a sixth aspect the invention provides a method for treatingcobalamin deficiency comprising administration of a composition thatincludes at least one cobalamin binding protein of the presentinvention. The deficiency can be overcome by providing additionalamounts of the proteins involved in vitamin B12 uptake and transport.These proteins are preferably any one or more of transcobalamin,haptocorrin and intrinsic factor. The proteins can be administered alonei.e. in the apo-form, or in conjunction with cobalamin or a cobalaminanalog i.e. in the holo-form. A “holo-fomm” of a cobalamin bindingprotein is a complex of the cobalamin binding protein and its ligande.g. cobalamin or a cobalamin analog. An “apo-form” of a cobalaminbinding protein is the cobalamin binding protein which is ready to binda cobalamin or a cobalamin analog.

[0033] In a seventh aspect the invention provides CBP of the inventionfor use in diagnostic tests. In such tests the content of cobalamins infor example blood is detected by the ability of the extracted biologicalsample to compete with tracer Cbl for binding to CBP as described byNexø and Gimsing, 1981, Scand. J. Clin. Lab. Invest. 41:465-468,:“Insolubilized pure human intrinsic factor used for quantitation ofcobalamins in serum”. In other tests the purpose is to measure the CBP,either its total content or its holo- or apo-form. Such tests can beperformed by for example enzyme linked immunosorbent assay (ELISA)directly (Nexø et al. 2000, Clinical Chemistry 46 (10):1643-1649), or incombination with a pretreatment of the sample that removes the apo-formi.e as described by Nexø, 1975, Biochim. Biophys. Acta, 379:189-192.

[0034] In another aspect the present invention provides a method forquantifying cobalamin absorption from the intestine by means of aSchilling test utilizing IF or a functional fragment thereof that hasbeen isolated from a transgenic plant expressing the cobalamin bindingprotein or a functional fragment thereof. For example, a “Schillingtest” may be performed by using a dose of Cbl with radioactive cobaltfor oral ingestion followed by 24 hours of urine collection andmeasurement of the fraction of radioactive Cbl in the urine. If noradioactive Cbl is detected in the urine of this first stage of thetest, a second test is performed a week later. Now the test uses a doseof Cbl with radioactive cobalt together with IF for oral ingestionfollowed by 24 hours of urine collection and measurement of the fractionof radioactive Cbl in the urine (Fairbanks, V. F. Test for perniciousanaemia: the “Schilling test” Mayo Clin Proc. 1983, 58: 541-544).

[0035] In a further aspect the invention provides a method forquantifying cobalamin or analogs thereof utilizing the protein of thepresent invention. The isolated CBP produced by the transgenic plant canbe used for example in a competitive binding assay utilizing labeledcobalamin or analogues thereof.

[0036] In yet a further aspect the present invention provides a methodfor quantifying CBP utilizing the protein of the present invention. TheCBP of the present invention can be isolated and then labeledradioactively, and used in a competitive binding assay with cobalamin oran analog thereof.

[0037] In yet another aspect the present invention provides a method forquantifying receptors for cobalamin binding proteins utilizing theprotein of the present invention. For example the rhIF may be complexedwith a labeled cobalamin molecule e.g. radioactive cobalmin, todetermine the number or binding capacity of the intestinal IF-B12receptor.

[0038] In a further aspect the present invention provides a nucleic acidconstruct comprising nucleic acid coding for one or more cobalaminbinding proteins, operably linked to one or more regulatory sequencescapable of directing expression in a plant. The regulatory sequence can,for example, be a promoter such as 35S CaMV. In addition a signalpeptide sequence such as the Arabidopsis thaliana extensin signalpeptide, Phalseolus vulgaris chitinase signal peptide, or Nicotianatabacum glucan beta-1,3-glucanase can also be included. In one preferredembodiment the nucleic acid is DNA.

[0039] In yet another aspect the present invention provides a vectorcomprising a nucleic acid construct of the invention. The vector may bea plasmid, cosmid or phage. Vectors frequently include one or moreexpressed markers which enable selection of cells transfected, ortransformed, with them and preferably, to enable a selection of cells,containing vectors incorporating heterologous DNA. If the vector isintended for expression, sufficient regulatory sequences to driveexpression will be present. The nucleic acid and promoter sequencesaccording to the invention are preferably for expression in plant cells.

[0040] In yet a further aspect the invention also provides a plant cellcomprising a nucleic acid sequence of the invention and/or a vector ofthe invention. The cell may be termed as a “host” which is useful formanipulation of the nucleic acid, including cloning. Alternatively, thecell may be a cell in which expression of the nucleic acid is obtained,most preferably a plant cell. The nucleic acid can be incorporated intocells by standard techniques known in the art. Preferably, nucleic acidis transformed into plant cells using a disarmed Ti plasmid vectorcarried in agrobacterium by procedures known in the art, for example, asdescribed in EP-A-0116718 and EP-A-0270822. Foreign nucleic acid canalternatively be introduced directly into plant cells using anelectrical discharge apparatus or by any other method that provides forthe stable incorporation of the nucleic acid into the cell. Nucleic acidof the present invention preferably contains a second “marker” gene thatenables identification of the nucleic acid. This is most commonly usedto distinguish the transformed plant cells containing the foreignnucleic acid from other plant cells that do not contain the foreignnucleic acid. Examples of such marker genes include antibioticresistance, herbicide resistance and glucoronidase (GUS) expression.Expression of the marker gene can be controlled by a second promoter,which allows expression of the marker gene in all cells.

[0041] The foreign nucleic acid is preferably introduced into only themitochondria and/or the chloroplasts of the host cell. The nucleic acidmay be become introduced into the genome, within the nucleus, or inother organelles, including mitochondria or plastids such asprotoplastids, chromoplasts or leucoplasts. Pollen does not containplastids or mitochondria. Therefore, by only having the foreign nucleicacids within these organelles, this will prevent the transfer oftransgenic material to wild type plants in the surrounding environmentby cross pollenation.

[0042] The aim of the present invention is to produce recombinantcobalamin binding proteins (CBP) in plants. Plants do not contain or usecobalamin and therefore the recombinant CBP expressed in plants will allbe in the apo-form (not complexed with cobalamin) whereas CBP fromnatural sources are a mixture of holo- and apo-form CBP. Unlike use oftransgenic plants, production of a particular CBP from natural sourcesinvolves further purification from other CBPs. Furthermore, transgenicplants provide a cheap method for large scale production of CBP, ascompared to all other known sources of the proteins.

[0043] The present invention will be easy to scale-up for production oflarge quantities of recombinant CBP by simply planting larger areas ofland with the CBP producing plants. This is relatively cheap compared toproduction of CBP in high technology demanding fermentors and incubatorswhen yeast or insect cells are used.

[0044] The use of natural sources to isolate CBP has some limitations.Because of the relatively low concentrations of TC in blood and milkthis protein is very expensive for isolation in large scale. Isolationof IF is performed from porcine stomach in large scale and to a lesserextent from rat stomach and human stomach juice. Since HC is alsopresent in the stomach, a high degree of purification is needed toseparate these CBP when IF is used for diagnostic kits. For example, IFfree of Cbl is requested (FDA requirement) for use as a binding proteinin assays of Cbl in plasma. In addition to these problems naturalsources also constitute a serious risk for transmission of diseases topatients receiving the CBP. Expression of recombinant CBP in transgenicplants will result in extracts with no other CBP than the recombinantCBP. Furthermore the risk of transmitting animal or human diseases fromnatural sources of CBP to patients is eliminated by the use of CBP fromtransgenic plants. Plant expression systems also give the opportunity touse GRAS (Generally Recognized As Safe) organisms as host for theproduction of CBP.

[0045] As examples of the use of transgenic plants as an expressionsystem for production of CBPs, we have expressed human IF and human TCin the plant Arabidopsis thaliana. We tested the transgenic plants forcobalamin binding capacity (CBC) as described by (14).

[0046] The description of the invention hereafter refers to Arabidopsisthialiana, when necessary for the sake of example. However, it should benoted that the invention is not limited to genetic transformation ofplants such as Arabidopsis thaliana. The method of the present inventionis capable of being practiced for other plant species, usingmethods/techniques well known to those skilled in the art.

[0047] The invention will now be described with reference to thefollowing examples, which should not be construed as in any way limitingthe invention.

[0048] The examples refer to the figures in which:

[0049]FIG. 1 shows the nucleotide sequence encoding extensin signalpeptide sequence fused to the mature human intrinsic factor encodingregion and part of the 3′-untranslated region. This nucleotide sequenceis a fusion of a nucleotide sequence (position 7-119) adopted fromGenBank accession no. AF104327 which encodes an Arabidopsis thalianaextensin-like signal peptide with the amino acid sequenceMASSSIALFLALNLLFFTISA. The methionine start codon (ATG) of this signalpeptide sequence is underlined. The nucleotide sequence encoding maturehuman intrinsic factor (position 120-1316) is shown in bold letters andadopted from GenBank accession no X76562. This sequence is followed by atranslational stop codon (TAA) which is underlined and a nucleotidesequence from the 3′-untranslated region of the intrinsic factor mRNA.The underlined restriction sites XbaI (position 1-6) and XmaI (position1425-1430) was introduced to facilitate cloning in the planttransformation vector.

[0050]FIG. 2 shows the amino acid sequence of mature human intrinsicfactor which was encoded by the nucleotide sequence shown in boldletters in FIG. 1 at position 120-1316.

[0051]FIG. 3 shows a nucleotide sequence at position 7-129 fromPhaseolus vulgaris CH5B-chitinase, GenBank accession no. S43926 and itencodes a signal peptide with the amino acid sequenceMKKNRMMIMICSVGVVWMLLVGGSYG. This nucleotide sequence was fused to thenucleotide sequence shown in bold letters in FIG. 1 that encodes themature human intrinsic factor. The XbaI restriction site used forcloning is underlined at position 1-6 and the translational start codonATG is underlined at position 52-54.

[0052]FIG. 4 shows a nucleotide sequence at position 7-144 fromNicotiana tabacum glucan beta-1,3-glucanase gene, GenBank accession no.M60402 and it encodes a signal peptide with the amino acid sequenceMSTSHKHNTPQMAAITLLGLLLVASSIDIAGA. This nucleotide sequence was fused tothe nucleotide sequence shown in bold letters in FIG. 1 that encodes themature human intrinsic factor. The XbaI restriction site is underlined(position 1-6) and the translational start codon ATG is underlined atposition 49-51.

[0053]FIG. 5 shows the nucleotide sequence encoding the extensin signalpeptide sequence fused to the mature human transcobalamin encodingregion. This nucleotide sequence is a fusion of a nucleotide sequence(position 7-119) adopted from GenBank accession no. AF104327 whichencodes an Arabidopsis thaliana extensin-like signal peptide with theamino acid sequence MASSSLFLFLALNLLFFTTISA. The methionine start codon(ATG) of this signal peptide sequence is underlined. The nucleotidesequence encoding mature human transcobalamin (position 120-1346) isshown in bold letters and adopted from GenBank accession noNM_(—)000355. This sequence is followed by a translational stop codon(TAG) at position 1347-1349 which is underlined. The underlinedrestriction sites XbaI (position 1-6) and XmaI (position 1350-1355) wasintroduced to facilitate cloning in the plant transformation vector.

[0054]FIG. 6 shows the amino acid sequence of mature humantranscobalamin which was encoded by the nucleotide sequence shown inbold letters in FIG. 5 at position 120-1346.

[0055]FIG. 7 shows the native intrinsic factor (∘) present in humangastric juice and recombinant human intrinsic factor extracted fromtransgenic plants of Arabidopsis thaliana () which were comparedconcerning their ability to bind cobalamin or cobinamid. Equal amountsof the respective proteins were added to a mixture containing a fixedamount of cobalt (57Co) labelled cobalamin mixed with increasing amountsof non-radioactive cobalamin or cobinamide (X axis). Free and boundligand were separated and the amount of 57Co attached to the protein wasmeasured in a gamma counter. The 57Co fraction bound relative to theamount of 57Co bound in the absence of unlabeled cobalamin or cobinamidewas calculated (Y axis). The figure shows that recombinant IF behaves asdoes native IF with specificity for binding cobalamin but not cobinamid.

[0056]FIG. 8 shows how polyclonal antibodies against human gastricintrinsic factor and alkaline phosphatase-conjugated immunoglobulinswere used to visualize intrinsic factor on a Western blot containingproteins separated by SDS-PAGE. Transgenic Arabidopsis thaliana plantsexpressing recombinant human intrinsic factor were harvested andhomogenized in 0.2 M phosphate buffer before centrifugation and analysisof the supernatant. For comparison transgenic yeast (Pichia pastoris)containing an insert of human intrinsic factor was used for expressionof recombinant human intrinsic factor. Secreted proteins form thefermentation media were precipitated with ammonium sulfate (80% w/v)before dialysis against 20 mM Tris pH 8.0 and analysis. Purified humangastric intrinsic factor was also used for comparison. An aliquot ofeach sample was treated with the enzyme PNGaseF to remove carbohydratefrom the proteins. Lane: “Plant IF” contains the proteins from the plantextract; “Plant IF+PNGaseF” contains the proteins from the plant extracttreated with PNGaseF; “Yeast IF” contains yeast protein; “YeastIF+PNGaseF contains yeast protein treated with PNGaseF; “Human gastricIF” contains purified human IF and “Human gastric IF+PNGaseF” containspurified human IF treated with PNGaseF. The arrow marks the 45 kDa bandwith deglycosylated IF from the three samples treated with PNGaseF.

[0057] The blot shows that the glycosylated form of IF from the threesamples have significant differences in molecular weight but thedeglycosylated samples contain mature IF with the same molecular weight.

[0058]FIG. 9 compares the glycosylation and molecular weight ofintrisicic factor prepared from various sources. PAS staining was usedto visualize glycoproteins. Recombinant human intrinsic factor (rhIF)from transgenic plants (Arabidopsis thaliana) and transgenic yeast(Pichia pastoris) were isolated and purified by affinity chromatrographyon a column with cobalamin. Purified human gastric IF was used forcomparison. Bovine PAS-3 is heavily glycosylated and function as acontrol for PAS staining. Bovine serum albumin (BSA) has noglycosylation and function as a “negative” control for PAS staining. Thesamples were run in SDS-PAGE in duplicate gels. The second gel wasstained with coomassie brilliant blue.

[0059] The gels show that recombinant plant IF is glycosylated since anapproximately 50 kDa rhIF band from transgenic plants is stained withPAS.

[0060]FIG. 10 shows the purification method for isolating rhIF and ablot of the protein fragments used for N-terminal sequencing. Purifiedrecombinant human intrinsic factor (rhIF) was run by SDS-PAGE, blottedonto a PVDF-membrane and stained with coomassie brilliant blue. Themature rhIF and the partially cleaved rhIF fragments (proteases presentin Arabidopsis thaliana cleave rhIF) were analyzed by amino acidsequencing. N-terminal sequensing of the three fragmens marked witharrows showed that the approximately 50 kDa fragment has the sameN-terminus as mature human gastric intrinsic factor. The lower twofragments (30 and 20 kDa) are a result of a proteolytic split in frontof amino acid residue 285 of mature hIF.

[0061]FIG. 11 shows the expression of transcobalamin in transgenicplants. Polyclonal antibodies against human transcobalamin (TC) andalkaline phosphatase-conjugated immunoglobulins were used to visualizeTC on a Western blot containing proteins separated by SDS-PAGE.Transgenic Arabidopsis thaliana plants (no 9-11) expressing recombinanthuman TC were harvested and homogenized in 0.2 M phosphate buffer beforecentrifugation and analysis of the supernatant.

[0062] The blot shows a single protein with the expected molecular sizeof 45 kDa in each of the three plants

[0063]FIG. 12 shows the ability of plant recombinant humantranscobalamin (∘) and plant recombinant human intrinsic factor () tobind cobalamin or cobinamid. Both recombinant proteins were extractedform transgenic Arabidopsis thaliana plants. Equal amounts of therespective proteins were added to a mixture containing a fixed amount ofcobalt (57Co) labeled cobalamin mixed with increasing amounts ofnon-radioactive cobalamin or cobinamide (X axis). Free and bound ligandwas separated and the amount of 57Co attached to the protein wasmeasured in a gamma counter. The 57Co fraction bound relative to theamount of 57Co bound in the absence of unlabelled cobalamin orcobinamide was calculated (Y axis). The figure shows that recombinanthuman intrinsic factor and recombinant human transcobalamin both bindcobalamin but not cobinamid.

[0064]FIG. 13 shows the binding of intrinsic factor to the humanintestinal receptor protein cubilin. The binding between intrinsicfactor and its receptor cubilin was analysed employing a BIAcore 2000instrument (Biacore AB, Uppsala, Sweden). An increase in Resp. Diff. (Yaxis) indicates binding between intrinsic factor and cubilin. At timeapproximately 100 a solution with intrinsic factor is added to theimmobilized cubilin. Intrinsic factor was isolated from transgenicplants, human gastric juice and hog stomach. Preparations of vitamin B12saturated and vitamin B12 unsaturated intrinsic factor were used. Attime approximately 600 intrinsic factor is removed from the solution andthe dissociation between cubilin and intrinsic factor is followed. Theresults indicate that recombinant intrinsic factor—like native intrinsicfactor—binds to cubilin only when saturated with vitamin B12 and thatthe binding characteristics for recombinant and native intrinsic factorare alike.

EXAMPLE 1

[0065] As shown in FIG. 1, the extensin signal peptide encodingnucleotide sequence from Arabidopsis thaliana was fused to thenucleotides encoding mature human intrinsic factor (FIG. 2). Thisconstruct was inserted in the plant transformation vector CRC-179.

[0066] Construction of the CRC-179 Vector

[0067] The vector pPZP 211 (Hajdukiewicz, P; Svab, Z; & Maliga, P. 1994Plant Mol. Biol. 25, 989-994) was digested with EcORI and KpnI and apAnos sequence was released from pGPTV KAN (Becker, D; Kemper, E;Schell, J & Masterson, R. 1992 Plant Mol Biol 20, 1195-1197) by the sameset of enzymes and cloned into the pPZP 211 vector. The resulting vectorwas digested with PstI and KpnI and blunt-ended. A blunt-ended EcORI andHindIII fragment containing the 35S CaMV promoter from the vectordescribed in Jefferson, R A; Kavanagh, T A & Bevan, M W. 1987 EMBO J. 6,3901-3907 was cloned into the blunt-ended vector. This vector was namedCRC-179. The bacteria Agrobacterium tumefaciens was transformed withthis recombinant vector.

[0068] Culture of Agrobacterium tumefaciens

[0069] The Agrobacterium tumefaciens strain used was GV3101 (pMP90)(Koncz and Schell, 1986) carrying the binary plasmid CRC-179 with aninsert encoding a CBP cloned into the XbaI-XmaI sites. The insertsequences are shown in the figures: 1, 3, 4, & 5. The bacteria weregrown to stationary phase in 200 ml liquid culture at 28-30° C., 250 rpmin sterilized LB media (10 g tryptone, 5 g yeast extract, 5 g NaCl perliter water) carrying added rifampicin (100 mg/ml), streptomycin (100mg/ml) and gentamycin (50 mg/ml). Cultures were started from a 1:200dilution of a smaller overnight culture and grown for 16-18 hours.Bacteria cells were harvested by centrifugation for 10 min at 5500 g atroom temperature and then resuspended in 400 ml inoculation medium (10mM MgCl₂, 5% w/v sucrose and 0.05% v/v Silwet L-77 (Lehle Seeds, RoundRock, Tex., USA)).

[0070] The recombinant A. tumefaciens bacteria were used to transformArabidopsis thaliana plants.

[0071] Transformation of Arabidopsis Plants

[0072] The Arabidopsis plants were transformed by the floral dip method(Clough and Bent, 1998).

[0073] Plant Growth

[0074]Arabidopsis plants (ecotype Col-0) were grown to flowering stagein growth chamber, 20° C. day/18° C. night with LiCl lighting for 18h/day, humidity 70%. Between 20 and 25 plants were planted per 64 cm²pot in moistened soil mixture consisting of: 40 kg soil orange and 40 kgsoil green (Stenrøgel Mosebrug A/S Kjellerup, DK), 25 liter 4-8 mmFibroklinker (Optiroc, Randers, DK), 12 liter Vermiculite (Skarnol, DK)and 300 g Osmocote plus NPK 15-5-11, 3-4 months (Scott's, UK).

[0075] To obtain more floral buds per plant, inflorescences were clippedafter most plants had formed primary bolts, relieving apical dominanceand encouraging synchronized emergence of multiple secondary bolts.Plants were dipped when most secondary inflorescences were about 7-13 cmtall (7-9 days after clipping).

[0076] The transgenic Agrobacterium suspension was added to a 400 mlbeaker and plants were inverted into the suspension such that allabove-ground tissues minus the rosette were submerged. The plants wereremoved after 10-15 sec of gentle agitation and placed in horizontalposition in a sealed plastic bag for 24 hours at room temperature. After24 hours the plants were moved to the growth camber and the plastic bagswere removed. Plants were grown 3-4 weeks until siliques were brown anddry. Seeds were harvested and allowed to dry at room temperature for 7days.

[0077] Selection of Transformants

[0078] Seed were surface sterilized by a treatment with 0.5% sodiumhypochlorite containing 0.05% v/v Tween 20 for 7 min, then with 70%ethanol for 2 min, followed by three rinses with sterile water.

[0079] To select for transformed plants the sterilized seeds were platedon kanamycin selection plates at a density of approximately 2000 seedsper 144 cm² and grown for 8-10 days at 21° C. under light for 16 hoursper day. Selection plates contained 1×MS medium (Duchefa, Haarlem, NL #M0222), 1% w/v sucrose, 0.9% w/v agar noble (Difco, Detroit, USA), 50mg/ml kanamycin, pH 5.7. After selection the transformed plants weretransferred to growth chambers (see Plant growth).

[0080] Seeds from these infected plants were planted and recombinantplants were identified by western-blotting analysis. Seeds fromrecombinant plants were used to grow new recombinant plants calledIF-plants.

[0081] One kilogram of three week old IF-plants was homogenization with2 liters of phosphate buffer and clarified by centrifugation. Thisextract contained 100 mg recombinant human IF with CBC. This IF hadspecificity for cobalamin binding whereas the analog cobinamid was notbound by the IF as tested by the method described by (15). FIG. 7 showsthat the rhIF from plants and native human gastric IF have the samespecificity for binding Cbl, but not cobinamid.

[0082] The transgenic plants were analyzed as described by (16) andshown to contain no cobalamin. This shows that the plant rhIF was at theapo-form. Analysis of the protein from these transgenic plants showedthat they express a protein of approximately 50 kDa as recognized on aWestern-blot with antibodies against human IF (see FIG. 8,10). Aminoacid sequencing of the N-terminal region of this 50 kDa protein showedthe same (FIG. 4). This construct was used to generate transgenicArabidopsis thaliana plants. These plants were shown to contain CBC atthe same level as most of the extensin-IF plants showing that the choiceof signal peptide for intrinsic factor expression is not restricted toone sequence.

[0083] Conclusions from IF-Plants in Examples 1-3

[0084] As far as we have tested the recombinant human IF from plants itbehaves as natural human gastric IF concerning its mature N-terminus,recognition by anti-IF antibodies, binding of cobalamin, lack ofcobinamide binding, binding to the intestinal receptor, and presence ofcarbohydrates. In contrast to gastric juice where IF is present togetherwith another CBP, haptocorrin, and to some extent cobalamin from thefood, transgenic IF-plants contain no cobalamin or other CBP than IF.The glycosylation of IF from transgenic plants was different from humangastric IF. Another difference between IF from human gastric mucosa andIF from transgenic plants is that IF from plants is at the apo-formwhereas IF from human beings is a mixture of the apo- and holo-form.

Example 4 TC-Plants

[0085] Extracts from one kilogram of transgenic Arabidopsis thaliana(TC-plants) transformed with an extensin-transcobalamin construct (FIG.5) contained 20 mg of recombinant human TC with CBC. Western blotanalysis showed a single band of approximately 45 kDa which reacted withantibodies against human TC (FIG. 11). The calculated molecular weightof mature TC (FIG. 6) with 409 amino acid residues is 45536 Da, showingthat the observed and calculated molecular weights are similar. Theseresults show that a plant expression system is able to producerecombinant human TC of the expected size and with CBC.

[0086] The transgenic plants were analyzed as described by (16) andshown to contain no cobalamin. This shows that the recombinant human TCwas at the apo-form. As for rhIF, rhTC obtained from plants binds to Cblbut not cobinamid (FIG. 12). N-terminal amino acid sequencing showedthat the extensin signal peptide was removed from the secreted rhTCgenertaing the normal mature N-terminal found in native humantranscobalamin.

Example 5 Purification of the Recombinant Intrinsic Factor from Plants

[0087] 1 kg of the crude plant material was chopped and homogenized in 2L of 0.2 M Sodium Phosphate buffer, pH 7.5. The homogenate wascentrifuged at 4000 rpm for 10 min and filtered through Watman paper ona Buchner funnel. The filtrate can be stored frozen at that stage.Intrinsic factor was adsorbed from the solution on an affinity matrixaccording to a previously described method (Nexø, E., 1975 Biochim.Biophys. Acta 379, 189-192). After elution from the column intrinsicfactor (saturated with cobalamin=holo-form) was subjected to gelfiltration, dialyzed against water and lyophilized. Preparation ofcobalamin unsaturated intrinsic factor (=apo-form) required anadditional step: dialysis against 5 M guanidine HCl for two daysfollowed by dialysis against 0.2 M Sodium Phosphate buffer, pH 7.5.

REFERENCES

[0088] 1 Allen, R. H. (1975) Prog. Hematol. 9, 57-84.

[0089] 2 Rothenberg, S. P., and Quadros, E. V. (1995) Bailliere S Clin.Haematol. 8, 499-514.

[0090] 3 Nicolas, J. P., and Gueant, J. L. (1995) Bailliere S Clin.Haematol. 8, 515-531.

[0091] 4 Nexø, E. (1998) in Vitamin B₁₂ and B₁₂-Proteins (Krautler, B.,Angoni, D., and Golding, B. T., eds) pp. 461-475, Wiley-VCH, Weinheim,Germany.

[0092] 5 Hall, C. A., and Begley, J. A. (1977) Am. J. Hum. Genet. 29,619-626.

[0093] 6 Katz, M., Mehlman, C. S., and Allen, R. H. (1974) J. Clin.Invest. 53, 1274-1283.

[0094] 7 Li, N., Rosenblatt, D. S., Kamen, B. A., Seetharam, S., andSeetharam, B. (1994) Hum, Mol, Genet. 3, 1835-1840.

[0095] 8 VanKapel, J., Loef, B. G., Lindemans, J., and Abels, J. (1981)Biochim, Biophys. Acta 676, 307-313.

[0096] 9 Quadros, E. V., Rothenberg, S. P., Pan, Y. C., and Stein, S.(1986) J. Biol. Chem. 261, 15455-15460.

[0097] 10 Gordon, M., Chokshi, H., and Alpers, D. H. (1992) Biochim.Biophys. Acta 1132, 276-283.

[0098] 11 Quadros, E. V., Sai, P., and Rothenberg, S. P. (1993) Blood81, 1239-1245.

[0099] 12 Fedosov, S. N., Berglund, L., Nexø, E., and Petersen, T. E.(1999) J. Biol. Chem. 274, 26015-26020.

[0100] 13 Wen, J., Kinnear, M. B., Richardson, M. A., Willetts, N. S.,Russell-Jones, G. J., Gordon, M. M., and Alpers, D. H. (2000) Biochim.Biophys. Acta 1490,43-53.

[0101] 14 Nexø, E. and Andersen, J. (1977) Scand. J. Clin. Lab. Invest.37, 723-728.

[0102] 15 Stupperich, E. And Nexø, E. (1991) Eur. J. Biochem. 199,299-303.

[0103] 16 Nexø, E. And Gimsing, P. (1981) Scand. J. Clin. Lab. Invest.41, 465-468.

[0104] 17 Clough, S. J., and Bent, A. F. (1998) Plant J. 16, 735-743.

[0105] 18 Koncz, C., and Schell, J. (1986) Mol. Gen. Genet. 204,383-396.

[0106] 19 Nexø et al. 2000, Clinical Chemistry 46 (10):1643-1649

[0107] 20 Nexø, 1975, Biochim. Biophys. Acta, 379:189-192.

1 11 1 22 PRT Arabidopsis thaliana 1 Met Ala Ser Ser Ser Ile Ala Leu PheLeu Ala Leu Asn Leu Leu Phe 1 5 10 15 Phe Thr Thr Ile Ser Ala 20 2 1430DNA Artificial extensin-IF fusion protein 2 tctagaactc acaacctagctagctagtaa acagtatttt ctatatacca aaa atg 56 Met 1 gct tca agt tcc atagct ctt ttc ttg gct ctc aat ctt ctc ttt ttc 104 Ala Ser Ser Ser Ile AlaLeu Phe Leu Ala Leu Asn Leu Leu Phe Phe 5 10 15 aca aca atc tcc gcc agtacc cag acc cag agt tca tgc tcc gtt ccc 152 Thr Thr Ile Ser Ala Ser ThrGln Thr Gln Ser Ser Cys Ser Val Pro 20 25 30 tca gca cag gag ccc ttg gtcaat gga ata caa gta ctc atg gag aac 200 Ser Ala Gln Glu Pro Leu Val AsnGly Ile Gln Val Leu Met Glu Asn 35 40 45 tcg gtg act tca tca gcc tac ccaaac ccc agc atc ctg att gcc atg 248 Ser Val Thr Ser Ser Ala Tyr Pro AsnPro Ser Ile Leu Ile Ala Met 50 55 60 65 aat ctg gcc gga gcc tac aac ttgaag gcc cag aag ctc ctg act tac 296 Asn Leu Ala Gly Ala Tyr Asn Leu LysAla Gln Lys Leu Leu Thr Tyr 70 75 80 cag ctc atg tcc agc gac aac aac gatcta acc att ggg cag ctc ggc 344 Gln Leu Met Ser Ser Asp Asn Asn Asp LeuThr Ile Gly Gln Leu Gly 85 90 95 ctc acc atc atg gcc ctc acc tcc tcc tgccga gac cct ggg gat aaa 392 Leu Thr Ile Met Ala Leu Thr Ser Ser Cys ArgAsp Pro Gly Asp Lys 100 105 110 gta tcc att cta caa aga caa atg gag aactgg gca cct tcc agc ccc 440 Val Ser Ile Leu Gln Arg Gln Met Glu Asn TrpAla Pro Ser Ser Pro 115 120 125 aac gct gaa gca tca gcc ttc tat ggg cccagt cta gcg atc ttg gca 488 Asn Ala Glu Ala Ser Ala Phe Tyr Gly Pro SerLeu Ala Ile Leu Ala 130 135 140 145 ctg tgc cag aag aac tct gag gcg accttg ccg ata gcc gtc cgc ttt 536 Leu Cys Gln Lys Asn Ser Glu Ala Thr LeuPro Ile Ala Val Arg Phe 150 155 160 gcc aag acc ctg ctg gcc aac tcc tctccc ttc aat gta gac aca gga 584 Ala Lys Thr Leu Leu Ala Asn Ser Ser ProPhe Asn Val Asp Thr Gly 165 170 175 gca atg gca acc ttg gct ctg acc tgtatg tac aac aag atc cct gta 632 Ala Met Ala Thr Leu Ala Leu Thr Cys MetTyr Asn Lys Ile Pro Val 180 185 190 ggt tca gag gaa ggt tac aga tcc ctgttt ggt cag gta cta aag gat 680 Gly Ser Glu Glu Gly Tyr Arg Ser Leu PheGly Gln Val Leu Lys Asp 195 200 205 att gtg gag aaa atc agc atg aag atcaaa gat aat ggc atc att gga 728 Ile Val Glu Lys Ile Ser Met Lys Ile LysAsp Asn Gly Ile Ile Gly 210 215 220 225 gac atc tac agt act ggc ctc gccatg cag gct ctc tct gta aca cct 776 Asp Ile Tyr Ser Thr Gly Leu Ala MetGln Ala Leu Ser Val Thr Pro 230 235 240 gag cca tct aaa aag gaa tgg aactgc aag aag act acg gat atg ata 824 Glu Pro Ser Lys Lys Glu Trp Asn CysLys Lys Thr Thr Asp Met Ile 245 250 255 ctc aat gag att aag cag ggg aaattc cac aac ccc atg tcc att gct 872 Leu Asn Glu Ile Lys Gln Gly Lys PheHis Asn Pro Met Ser Ile Ala 260 265 270 caa atc ctc cct tcc ctg aaa ggcaag aca tac cta gat gtg ccc cag 920 Gln Ile Leu Pro Ser Leu Lys Gly LysThr Tyr Leu Asp Val Pro Gln 275 280 285 gtc act tgt agt cct gat cat gaggta caa cca act cta ccc agc aac 968 Val Thr Cys Ser Pro Asp His Glu ValGln Pro Thr Leu Pro Ser Asn 290 295 300 305 cct ggc cct ggc ccc acc tctgca tct aac atc act gtc ata tac acc 1016 Pro Gly Pro Gly Pro Thr Ser AlaSer Asn Ile Thr Val Ile Tyr Thr 310 315 320 ata aat aac cag ctg agg ggggtt gag ctg ctc ttc aac gag acc atc 1064 Ile Asn Asn Gln Leu Arg Gly ValGlu Leu Leu Phe Asn Glu Thr Ile 325 330 335 aat gtt agt gtg aaa agt gggtca gtg tta ctt gtt gtc cta gag gaa 1112 Asn Val Ser Val Lys Ser Gly SerVal Leu Leu Val Val Leu Glu Glu 340 345 350 gca cag cgc aaa aat cct atgttc aaa ttt gaa acc aca atg aca tct 1160 Ala Gln Arg Lys Asn Pro Met PheLys Phe Glu Thr Thr Met Thr Ser 355 360 365 tgg ggc ctt gtc gtc tct tctatc aac aat atc gcg gaa aat gtt aat 1208 Trp Gly Leu Val Val Ser Ser IleAsn Asn Ile Ala Glu Asn Val Asn 370 375 380 385 cac aag aca tac tgg cagttt ctt agt ggt gta aca cct ttg aat gaa 1256 His Lys Thr Tyr Trp Gln PheLeu Ser Gly Val Thr Pro Leu Asn Glu 390 395 400 ggg gtt gct gac tac ataccc ttc aac cac gag cac atc aca gcc aat 1304 Gly Val Ala Asp Tyr Ile ProPhe Asn His Glu His Ile Thr Ala Asn 405 410 415 ttc aca cag tactaacgaagag gtgggttcag cttctatcaa acatctccaa 1356 Phe Thr Gln Tyr 420aggatgggtg aaattttttc cacttcattt taaatctatg caaaaaagcg aatgcctgtg 1416atgctacccc cggg 1430 3 421 PRT Artificial extensin-IF fusion protein 3Met Ala Ser Ser Ser Ile Ala Leu Phe Leu Ala Leu Asn Leu Leu Phe 1 5 1015 Phe Thr Thr Ile Ser Ala Ser Thr Gln Thr Gln Ser Ser Cys Ser Val 20 2530 Pro Ser Ala Gln Glu Pro Leu Val Asn Gly Ile Gln Val Leu Met Glu 35 4045 Asn Ser Val Thr Ser Ser Ala Tyr Pro Asn Pro Ser Ile Leu Ile Ala 50 5560 Met Asn Leu Ala Gly Ala Tyr Asn Leu Lys Ala Gln Lys Leu Leu Thr 65 7075 80 Tyr Gln Leu Met Ser Ser Asp Asn Asn Asp Leu Thr Ile Gly Gln Leu 8590 95 Gly Leu Thr Ile Met Ala Leu Thr Ser Ser Cys Arg Asp Pro Gly Asp100 105 110 Lys Val Ser Ile Leu Gln Arg Gln Met Glu Asn Trp Ala Pro SerSer 115 120 125 Pro Asn Ala Glu Ala Ser Ala Phe Tyr Gly Pro Ser Leu AlaIle Leu 130 135 140 Ala Leu Cys Gln Lys Asn Ser Glu Ala Thr Leu Pro IleAla Val Arg 145 150 155 160 Phe Ala Lys Thr Leu Leu Ala Asn Ser Ser ProPhe Asn Val Asp Thr 165 170 175 Gly Ala Met Ala Thr Leu Ala Leu Thr CysMet Tyr Asn Lys Ile Pro 180 185 190 Val Gly Ser Glu Glu Gly Tyr Arg SerLeu Phe Gly Gln Val Leu Lys 195 200 205 Asp Ile Val Glu Lys Ile Ser MetLys Ile Lys Asp Asn Gly Ile Ile 210 215 220 Gly Asp Ile Tyr Ser Thr GlyLeu Ala Met Gln Ala Leu Ser Val Thr 225 230 235 240 Pro Glu Pro Ser LysLys Glu Trp Asn Cys Lys Lys Thr Thr Asp Met 245 250 255 Ile Leu Asn GluIle Lys Gln Gly Lys Phe His Asn Pro Met Ser Ile 260 265 270 Ala Gln IleLeu Pro Ser Leu Lys Gly Lys Thr Tyr Leu Asp Val Pro 275 280 285 Gln ValThr Cys Ser Pro Asp His Glu Val Gln Pro Thr Leu Pro Ser 290 295 300 AsnPro Gly Pro Gly Pro Thr Ser Ala Ser Asn Ile Thr Val Ile Tyr 305 310 315320 Thr Ile Asn Asn Gln Leu Arg Gly Val Glu Leu Leu Phe Asn Glu Thr 325330 335 Ile Asn Val Ser Val Lys Ser Gly Ser Val Leu Leu Val Val Leu Glu340 345 350 Glu Ala Gln Arg Lys Asn Pro Met Phe Lys Phe Glu Thr Thr MetThr 355 360 365 Ser Trp Gly Leu Val Val Ser Ser Ile Asn Asn Ile Ala GluAsn Val 370 375 380 Asn His Lys Thr Tyr Trp Gln Phe Leu Ser Gly Val ThrPro Leu Asn 385 390 395 400 Glu Gly Val Ala Asp Tyr Ile Pro Phe Asn HisGlu His Ile Thr Ala 405 410 415 Asn Phe Thr Gln Tyr 420 4 399 PRT Homosapiens 4 Ser Thr Gln Thr Gln Ser Ser Cys Ser Val Pro Ser Ala Gln GluPro 1 5 10 15 Leu Val Asn Gly Ile Gln Val Leu Met Glu Asn Ser Val ThrSer Ser 20 25 30 Ala Tyr Pro Asn Pro Ser Ile Leu Ile Ala Met Asn Leu AlaGly Ala 35 40 45 Tyr Asn Leu Lys Ala Gln Lys Leu Leu Thr Tyr Gln Leu MetSer Ser 50 55 60 Asp Asn Asn Asp Leu Thr Ile Gly Gln Leu Gly Leu Thr IleMet Ala 65 70 75 80 Leu Thr Ser Ser Cys Arg Asp Pro Gly Asp Lys Val SerIle Leu Gln 85 90 95 Arg Gln Met Glu Asn Trp Ala Pro Ser Ser Pro Asn AlaGlu Ala Ser 100 105 110 Ala Phe Tyr Gly Pro Ser Leu Ala Ile Leu Ala LeuCys Gln Lys Asn 115 120 125 Ser Glu Ala Thr Leu Pro Ile Ala Val Arg PheAla Lys Thr Leu Leu 130 135 140 Ala Asn Ser Ser Pro Phe Asn Val Asp ThrGly Ala Met Ala Thr Leu 145 150 155 160 Ala Leu Thr Cys Met Tyr Asn LysIle Pro Val Gly Ser Glu Glu Gly 165 170 175 Tyr Arg Ser Leu Phe Gly GlnVal Leu Lys Asp Ile Val Glu Lys Ile 180 185 190 Ser Met Lys Ile Lys AspAsn Gly Ile Ile Gly Asp Ile Tyr Ser Thr 195 200 205 Gly Leu Ala Met GlnAla Leu Ser Val Thr Pro Glu Pro Ser Lys Lys 210 215 220 Glu Trp Asn CysLys Lys Thr Thr Asp Met Ile Leu Asn Glu Ile Lys 225 230 235 240 Gln GlyLys Phe His Asn Pro Met Ser Ile Ala Gln Ile Leu Pro Ser 245 250 255 LeuLys Gly Lys Thr Tyr Leu Asp Val Pro Gln Val Thr Cys Ser Pro 260 265 270Asp His Glu Val Gln Pro Thr Leu Pro Ser Asn Pro Gly Pro Gly Pro 275 280285 Thr Ser Ala Ser Asn Ile Thr Val Ile Tyr Thr Ile Asn Asn Gln Leu 290295 300 Arg Gly Val Glu Leu Leu Phe Asn Glu Thr Ile Asn Val Ser Val Lys305 310 315 320 Ser Gly Ser Val Leu Leu Val Val Leu Glu Glu Ala Gln ArgLys Asn 325 330 335 Pro Met Phe Lys Phe Glu Thr Thr Met Thr Ser Trp GlyLeu Val Val 340 345 350 Ser Ser Ile Asn Asn Ile Ala Glu Asn Val Asn HisLys Thr Tyr Trp 355 360 365 Gln Phe Leu Ser Gly Val Thr Pro Leu Asn GluGly Val Ala Asp Tyr 370 375 380 Ile Pro Phe Asn His Glu His Ile Thr AlaAsn Phe Thr Gln Tyr 385 390 395 5 129 DNA Phaseolus vulgaris CDS(52)..(129) 5 tctagaagag agggaaggaa gggaaacgga aagagagaaa gagaaagaga aatg aag 57 Met Lys 1 aag aat agg atg atg att atg ata tgc agt gta gga gtggtg tgg atg 105 Lys Asn Arg Met Met Ile Met Ile Cys Ser Val Gly Val ValTrp Met 5 10 15 ctg tta gtt gga gga agc tac gga 129 Leu Leu Val Gly GlySer Tyr Gly 20 25 6 26 PRT Phaseolus vulgaris 6 Met Lys Lys Asn Arg MetMet Ile Met Ile Cys Ser Val Gly Val Val 1 5 10 15 Trp Met Leu Leu ValGly Gly Ser Tyr Gly 20 25 7 144 DNA Nicotiana tabacum CDS (49)..(144) 7tctagaagct cgttgttcat cttaattctc ccaacaagtc ttcccatc atg tct acc 57 MetSer Thr 1 tca cat aaa cat aat act cct caa atg gct gct atc aca ctc ctagga 105 Ser His Lys His Asn Thr Pro Gln Met Ala Ala Ile Thr Leu Leu Gly5 10 15 tta cta ctt gtt gcc agc agc att gac ata gca ggg gct 144 Leu LeuLeu Val Ala Ser Ser Ile Asp Ile Ala Gly Ala 20 25 30 8 32 PRT Nicotianatabacum 8 Met Ser Thr Ser His Lys His Asn Thr Pro Gln Met Ala Ala IleThr 1 5 10 15 Leu Leu Gly Leu Leu Leu Val Ala Ser Ser Ile Asp Ile AlaGly Ala 20 25 30 9 1355 DNA Artificial extensin-transcobalamin fusionprotein 9 tctagaactc acaacctagc tagctagtaa acagtatttt ctatatacca aaa atg56 Met 1 gct tca agt tcc ata gct ctt ttc ttg gct ctc aat ctt ctc ttt ttc104 Ala Ser Ser Ser Ile Ala Leu Phe Leu Ala Leu Asn Leu Leu Phe Phe 5 1015 aca aca atc tcc gcc gag atg tgt gaa ata cca gag atg gac agc cat 152Thr Thr Ile Ser Ala Glu Met Cys Glu Ile Pro Glu Met Asp Ser His 20 25 30ctg gta gag aag ttg ggc cag cac ctc tta cct tgg atg gac cgg ctt 200 LeuVal Glu Lys Leu Gly Gln His Leu Leu Pro Trp Met Asp Arg Leu 35 40 45 tccctg gag cac ttg aac ccc agc atc tat gtg ggc cta cgc ctc tcc 248 Ser LeuGlu His Leu Asn Pro Ser Ile Tyr Val Gly Leu Arg Leu Ser 50 55 60 65 agtctg cag gct ggg acc aag gaa gac ctc tac ctg cac agc ctc aag 296 Ser LeuGln Ala Gly Thr Lys Glu Asp Leu Tyr Leu His Ser Leu Lys 70 75 80 ctt ggttac cag cag tgc ctc cta ggg tct gcc ttc agc gag gat gac 344 Leu Gly TyrGln Gln Cys Leu Leu Gly Ser Ala Phe Ser Glu Asp Asp 85 90 95 ggt gac tgccag ggc aag cct tcc atg ggc cag ctg gcc ctc tac ctg 392 Gly Asp Cys GlnGly Lys Pro Ser Met Gly Gln Leu Ala Leu Tyr Leu 100 105 110 ctc gct ctcaga gcc aac tgt gag ttt gtc agg ggc cac aag ggg gac 440 Leu Ala Leu ArgAla Asn Cys Glu Phe Val Arg Gly His Lys Gly Asp 115 120 125 agg ctg gtctca cag ctc aaa tgg ttc ctg gag gat gag aag aga gcc 488 Arg Leu Val SerGln Leu Lys Trp Phe Leu Glu Asp Glu Lys Arg Ala 130 135 140 145 att gggcat gat cac aag ggc cac ccc cac act agc tac tac cag tat 536 Ile Gly HisAsp His Lys Gly His Pro His Thr Ser Tyr Tyr Gln Tyr 150 155 160 ggc ctgggc att ctg gcc ctg tgt ctc cac cag aag cgg gtc cat gac 584 Gly Leu GlyIle Leu Ala Leu Cys Leu His Gln Lys Arg Val His Asp 165 170 175 agc gtggtg gac aaa ctt ctg tat gct gtg gaa cct ttc cac cag ggc 632 Ser Val ValAsp Lys Leu Leu Tyr Ala Val Glu Pro Phe His Gln Gly 180 185 190 cac cattct gtg gac aca gca gcc atg gca ggc ttg gca ttc acc tgt 680 His His SerVal Asp Thr Ala Ala Met Ala Gly Leu Ala Phe Thr Cys 195 200 205 ctg aagcgc tca aac ttc aac cct ggt cgg aga caa cgg atc acc atg 728 Leu Lys ArgSer Asn Phe Asn Pro Gly Arg Arg Gln Arg Ile Thr Met 210 215 220 225 gccatc aga aca gtg cga gag gag atc ttg aag gcc cag acc ccc gag 776 Ala IleArg Thr Val Arg Glu Glu Ile Leu Lys Ala Gln Thr Pro Glu 230 235 240 ggccac ttt ggg aat gtc tac agc acc cca ttg gca tta cag ttc ctc 824 Gly HisPhe Gly Asn Val Tyr Ser Thr Pro Leu Ala Leu Gln Phe Leu 245 250 255 atgact tcc ccc atg cct ggg gca gaa ctg gga aca gca tgt ctc aag 872 Met ThrSer Pro Met Pro Gly Ala Glu Leu Gly Thr Ala Cys Leu Lys 260 265 270 gcgagg gtt gct ttg ctg gcc agt ctg cag gat gga gcc ttc cag aat 920 Ala ArgVal Ala Leu Leu Ala Ser Leu Gln Asp Gly Ala Phe Gln Asn 275 280 285 gctctc atg att tcc cag ctg ctg ccc gtt ctg aac cac aag acc tac 968 Ala LeuMet Ile Ser Gln Leu Leu Pro Val Leu Asn His Lys Thr Tyr 290 295 300 305att gat ctg atc ttc cca gac tgt ctg gca cca cga gtc atg ttg gaa 1016 IleAsp Leu Ile Phe Pro Asp Cys Leu Ala Pro Arg Val Met Leu Glu 310 315 320cca gct gct gag acc att cct cag acc caa gag atc atc agt gtc acg 1064 ProAla Ala Glu Thr Ile Pro Gln Thr Gln Glu Ile Ile Ser Val Thr 325 330 335ctg cag gtg ctt agt ctc ttg ccg ccg tac aga cag tcc atc tct gtt 1112 LeuGln Val Leu Ser Leu Leu Pro Pro Tyr Arg Gln Ser Ile Ser Val 340 345 350ctg gcc ggg tcc acc gtg gaa gat gtc ctg aag aag gcc cat gag tta 1160 LeuAla Gly Ser Thr Val Glu Asp Val Leu Lys Lys Ala His Glu Leu 355 360 365gga gga ttc aca tat gaa aca cag gcc tcc ttg tca ggc ccc tac tta 1208 GlyGly Phe Thr Tyr Glu Thr Gln Ala Ser Leu Ser Gly Pro Tyr Leu 370 375 380385 acc tcc gtg atg ggg aaa gcg gcc gga gaa agg gag ttc tgg cag ctt 1256Thr Ser Val Met Gly Lys Ala Ala Gly Glu Arg Glu Phe Trp Gln Leu 390 395400 ctc cga gac ccc aac acc cca ctg ttg caa ggt att gct gac tac aga 1304Leu Arg Asp Pro Asn Thr Pro Leu Leu Gln Gly Ile Ala Asp Tyr Arg 405 410415 ccc aag gat gga gaa acc att gag ctg agg ctg gtt agc tgg tagcccggg1355 Pro Lys Asp Gly Glu Thr Ile Glu Leu Arg Leu Val Ser Trp 420 425 43010 431 PRT Artificial extensin-transcobalamin fusion protein 10 Met AlaSer Ser Ser Ile Ala Leu Phe Leu Ala Leu Asn Leu Leu Phe 1 5 10 15 PheThr Thr Ile Ser Ala Glu Met Cys Glu Ile Pro Glu Met Asp Ser 20 25 30 HisLeu Val Glu Lys Leu Gly Gln His Leu Leu Pro Trp Met Asp Arg 35 40 45 LeuSer Leu Glu His Leu Asn Pro Ser Ile Tyr Val Gly Leu Arg Leu 50 55 60 SerSer Leu Gln Ala Gly Thr Lys Glu Asp Leu Tyr Leu His Ser Leu 65 70 75 80Lys Leu Gly Tyr Gln Gln Cys Leu Leu Gly Ser Ala Phe Ser Glu Asp 85 90 95Asp Gly Asp Cys Gln Gly Lys Pro Ser Met Gly Gln Leu Ala Leu Tyr 100 105110 Leu Leu Ala Leu Arg Ala Asn Cys Glu Phe Val Arg Gly His Lys Gly 115120 125 Asp Arg Leu Val Ser Gln Leu Lys Trp Phe Leu Glu Asp Glu Lys Arg130 135 140 Ala Ile Gly His Asp His Lys Gly His Pro His Thr Ser Tyr TyrGln 145 150 155 160 Tyr Gly Leu Gly Ile Leu Ala Leu Cys Leu His Gln LysArg Val His 165 170 175 Asp Ser Val Val Asp Lys Leu Leu Tyr Ala Val GluPro Phe His Gln 180 185 190 Gly His His Ser Val Asp Thr Ala Ala Met AlaGly Leu Ala Phe Thr 195 200 205 Cys Leu Lys Arg Ser Asn Phe Asn Pro GlyArg Arg Gln Arg Ile Thr 210 215 220 Met Ala Ile Arg Thr Val Arg Glu GluIle Leu Lys Ala Gln Thr Pro 225 230 235 240 Glu Gly His Phe Gly Asn ValTyr Ser Thr Pro Leu Ala Leu Gln Phe 245 250 255 Leu Met Thr Ser Pro MetPro Gly Ala Glu Leu Gly Thr Ala Cys Leu 260 265 270 Lys Ala Arg Val AlaLeu Leu Ala Ser Leu Gln Asp Gly Ala Phe Gln 275 280 285 Asn Ala Leu MetIle Ser Gln Leu Leu Pro Val Leu Asn His Lys Thr 290 295 300 Tyr Ile AspLeu Ile Phe Pro Asp Cys Leu Ala Pro Arg Val Met Leu 305 310 315 320 GluPro Ala Ala Glu Thr Ile Pro Gln Thr Gln Glu Ile Ile Ser Val 325 330 335Thr Leu Gln Val Leu Ser Leu Leu Pro Pro Tyr Arg Gln Ser Ile Ser 340 345350 Val Leu Ala Gly Ser Thr Val Glu Asp Val Leu Lys Lys Ala His Glu 355360 365 Leu Gly Gly Phe Thr Tyr Glu Thr Gln Ala Ser Leu Ser Gly Pro Tyr370 375 380 Leu Thr Ser Val Met Gly Lys Ala Ala Gly Glu Arg Glu Phe TrpGln 385 390 395 400 Leu Leu Arg Asp Pro Asn Thr Pro Leu Leu Gln Gly IleAla Asp Tyr 405 410 415 Arg Pro Lys Asp Gly Glu Thr Ile Glu Leu Arg LeuVal Ser Trp 420 425 430 11 409 PRT Homo sapiens 11 Glu Met Cys Glu IlePro Glu Met Asp Ser His Leu Val Glu Lys Leu 1 5 10 15 Gly Gln His LeuLeu Pro Trp Met Asp Arg Leu Ser Leu Glu His Leu 20 25 30 Asn Pro Ser IleTyr Val Gly Leu Arg Leu Ser Ser Leu Gln Ala Gly 35 40 45 Thr Lys Glu AspLeu Tyr Leu His Ser Leu Lys Leu Gly Tyr Gln Gln 50 55 60 Cys Leu Leu GlySer Ala Phe Ser Glu Asp Asp Gly Asp Cys Gln Gly 65 70 75 80 Lys Pro SerMet Gly Gln Leu Ala Leu Tyr Leu Leu Ala Leu Arg Ala 85 90 95 Asn Cys GluPhe Val Arg Gly His Lys Gly Asp Arg Leu Val Ser Gln 100 105 110 Leu LysTrp Phe Leu Glu Asp Glu Lys Arg Ala Ile Gly His Asp His 115 120 125 LysGly His Pro His Thr Ser Tyr Tyr Gln Tyr Gly Leu Gly Ile Leu 130 135 140Ala Leu Cys Leu His Gln Lys Arg Val His Asp Ser Val Val Asp Lys 145 150155 160 Leu Leu Tyr Ala Val Glu Pro Phe His Gln Gly His His Ser Val Asp165 170 175 Thr Ala Ala Met Ala Gly Leu Ala Phe Thr Cys Leu Lys Arg SerAsn 180 185 190 Phe Asn Pro Gly Arg Arg Gln Arg Ile Thr Met Ala Ile ArgThr Val 195 200 205 Arg Glu Glu Ile Leu Lys Ala Gln Thr Pro Glu Gly HisPhe Gly Asn 210 215 220 Val Tyr Ser Thr Pro Leu Ala Leu Gln Phe Leu MetThr Ser Pro Met 225 230 235 240 Pro Gly Ala Glu Leu Gly Thr Ala Cys LeuLys Ala Arg Val Ala Leu 245 250 255 Leu Ala Ser Leu Gln Asp Gly Ala PheGln Asn Ala Leu Met Ile Ser 260 265 270 Gln Leu Leu Pro Val Leu Asn HisLys Thr Tyr Ile Asp Leu Ile Phe 275 280 285 Pro Asp Cys Leu Ala Pro ArgVal Met Leu Glu Pro Ala Ala Glu Thr 290 295 300 Ile Pro Gln Thr Gln GluIle Ile Ser Val Thr Leu Gln Val Leu Ser 305 310 315 320 Leu Leu Pro ProTyr Arg Gln Ser Ile Ser Val Leu Ala Gly Ser Thr 325 330 335 Val Glu AspVal Leu Lys Lys Ala His Glu Leu Gly Gly Phe Thr Tyr 340 345 350 Glu ThrGln Ala Ser Leu Ser Gly Pro Tyr Leu Thr Ser Val Met Gly 355 360 365 LysAla Ala Gly Glu Arg Glu Phe Trp Gln Leu Leu Arg Asp Pro Asn 370 375 380Thr Pro Leu Leu Gln Gly Ile Ala Asp Tyr Arg Pro Lys Asp Gly Glu 385 390395 400 Thr Ile Glu Leu Arg Leu Val Ser Trp 405

1. A transgenic plant that expresses intrinsic factor or a protein withat least 60% sequence identity to intrinsic factor or a functionalfragment thereof capable of binding cobalamin and/or an analog thereof.2. The transgenic plant according to claim 1, wherein said protein is inthe apoform.
 3. The transgenic plant according to claim 1, wherein theplant is “Generally Recognized A Safe” (GRAS).
 4. The transgenic plantaccording to claim 1, wherein the plant is Arabidopsis thaliana. 5.Propagating material derived from a transgenic plant as defined inclaim
 1. 6. The propagating material according to claim 5, whichcomprises seeds.
 7. Intrinsic factor or a protein with at least 60%sequence identity to intrinsic factor or a functional fragment thereofthat is expressed in a transgenic plant and is capable of bindingcobalamin and/or an analog thereof.
 8. Intrinsic factor or a proteinwith at least 60% sequence identity to intrinsic factor or a functionalfragment thereof according to claim 7, which is isolated and/or purifiedfrom the transgenic plant.
 9. Intrinsic factor or a protein with atleast 60% sequence identity to intrinsic factor or a functional fragmentthereof according to claim 7, said protein being in the apoform.
 10. Amethod of isolating and/or purifying intrinsic factor or a protein withat least 60% sequence identity to intrinsic factor or a functionalfragment thereof as defined in claim 7, which includes the steps of:homogenizing the transgenic plant.
 11. The method according to claim 10,which comprises the following steps: filtration of the supernatantformed by centrifugation of the homogenate, affinity columnchromatography using cobalamin, elution of the cobalamin binding proteinattached to the cobalamin, gel filtration; and dialysis against water;and optionally further comprising the following steps to produce theapoform of the cobalamin: dialysis against 5M guanidine HCl; anddialysis against 0.2M sodium phosphate.
 12. A composition comprising acobalamin binding protein or a functional fragment thereof as defined inclaim
 7. 13. The composition according to claim 12, which is apharmaceutical composition and optionally further comprises one or morepharmaceutically acceptable diluents, excipients and/or carriers. 14.The composition according to claim 12, further comprising a secondcomponent selected from cobalamin, cobalamin analog or cobalamincontaining a tracer molecule.
 15. The composition according to claim 12which is for oral use.
 16. A foodstuff comprising transgenic plants asdefined in claim 1 or a plant material derived from said plant.
 17. Theuse of a protein as defined in claim 7 in diagnostic tests.
 18. A methodfor quantifying cobalamin absorption from the intestine by means of aSchilling test utilizing a protein as defined in claim 7, wherein theprotein is intrinsic factor or a functional fragment thereof.
 19. Amethod for quantifying a cobalamin binding protein utilizing a proteinas defined in claim
 7. 20. A method for quantifying a cobalamin bindingprotein utilizing a protein as defined in claim 7, or antibodies againstsaid protein.
 21. A method for quantifying receptors for a cobalaminbinding protein utilizing a protein as defined in claim
 7. 22. Themethod according to claim 21 further utilizing labelled cobalamin. 23.The method according to claim 22, wherein the cobalamin is radioactivelylabelled.
 24. A nucleic acid construct comprising nucleic acid codingfor intrinsic factor or a protein with at least 60% sequence identity tointrinsic factor or a functional fragment thereof, operably linked toone or more regulatory sequences capable of directing expression in aplant.
 25. The nucleic acid construct according to claim 24, wherein thenucleic acid is DNA.
 26. A nucleic acid according to claim 24, whereinthe regulatory sequences are selected from any one of Arabidopsisthaliana extensin signal peptide, Phalsoelus vulgaris chitinase signalpeptide, Nicotiana tabacum glucan beta-1,3-glucanase and/or 35Spromoter.
 27. A vector comprising a nucleic acid construct as defined inclaim
 24. 28. A plant cell comprising a vector as claimed in claim 27.29. A whole plant or part thereof, comprising a plant cell as defined inclaim
 28. 30. A method for treating cobalamin deficiency comprisingadministering a protein or a functional fragment thereof as defined inclaim 7 to a subject in need thereof.
 31. The method according to claim30, wherein the cobalamin deficiency is caused by low levels of at leastone of intrinsic factor, transcobalamin and haptocorrin.
 32. Cancel 33.Cancel